International Journal of Systematic and Evolutionary Microbiology (2011), 61, 493–496 DOI 10.1099/ijs.0.019810-0

Agarivorans gilvus sp. nov. isolated from seaweed

Zong-Jun Du,1,3 Guo-Qiang Lv,1 Alejandro P. Rooney,2 Ting-Ting Miao,1 Qing-Qiang Xu1 and Guan-Jun Chen1,3

Correspondence 1College of Marine Science, Shandong University at Weihai, Weihai 264209, PR China Guan-Jun Chen 2National Center for Agricultural Utilization Research, Agricultural Research Service, US [email protected] Department of Agriculture, 1815 North University Street, Peoria, IL 61604, USA 3State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, PR China

A novel agarase-producing, non-endospore-forming marine bacterium, WH0801T, was isolated from a fresh seaweed sample collected from the coast of Weihai, China. Preliminary characterization based on 16S rRNA gene sequence analysis showed that WH0801T shared 96.1 % similarity with Agarivorans albus MKT 106T, the type species of the genus Agarivorans.A polyphasic taxonomic study was conducted and confirmed the phylogenetic affiliation of strain WH0801T to the genus Agarivorans. Isolate WH0801T produces light-yellow-pigmented colonies; cells are Gram-stain-negative, straight or curved rods, which are motile with a single polar flagellum. Strain WH0801T grew in 0.5–5 % NaCl, with optimum growth at 3 % NaCl, and its optimal pH and cultivation temperature were 8.4–8.6 and 28–32 6C, respectively. Data from biochemical tests, whole-cell fatty acid profiling, 16S rRNA gene sequence studies and DNA– DNA hybridization clearly indicated that isolate WH0801T represented a novel species within the genus Agarivorans, for which the name Agarivorans gilvus sp. nov. is proposed. The type strain of Agarivorans gilvus sp. nov. is WH0801T (5NRRL B-59247T 5CGMCC 1.10131T).

The genus Agarivorans was created by Kurahashi & Yokota the egg jelly coating of certain sea urchin species, and the (2004) to accommodate Gram-negative, strictly aerobic outer tunics of ascidians (Pomin, 2010). We examined the and agar-hydrolysing species. The genus is affiliated with diversity of agarolytic marine bacteria isolated from various the class Gammaproteobacteria and presently contains only marine samples such as seawater, sediment, seaweed and the type species, Agarivorans albus. The Agarivorans albus some marine animal samples that were taken from waters strains studied by Kurahashi & Yokota were agarolytic and off various locations across coastal China. As a result of isolated from healthy marine organisms collected from these efforts, we have already described one new genus, the coast of the Kanto area in Japan. Many novel agar- Gilvimarinus (Du et al., 2009). Here, we report the taxono- degrading bacterial species have been isolated from marine mic characteristics of a novel species of the genus Agarivorans environments, where they occur as part of the indigenous that was isolated from seaweed sampled off the coast of flora. For example, Simiduia agarivorans, Aliagarivorans Weihai, China. marinus, Aliagarivorans taiwanensis and Tamlana agarivor- T ans were isolated from seawater samples (Shieh et al., 2008; Strain WH0801 was isolated from the surface of seaweed Jean et al., 2009; Yoon et al., 2008); Psychromonas collected from the shallow coastal region of Weihai, China. agarivorans, Microbulbifer agarilyticus and Marinimicrobium The seaweed samples were washed several times with sterile agarilyticum were found in marine sediment samples seawater and subsequently put into a centrifuge tube with (Hosoya et al., 2009; Miyazaki et al., 2008; Lim et al., sterile seawater and shaken vigorously. Aliquots (0.1 ml 2006); and Salegentibacter agarivorans was recovered from a each) of the dilution were spread onto marine agar 2216 species of sponge (Nedashkovskaya et al.,2006).The (MA; BD). Plates were then incubated at 25 uC for 3 to widespread occurrence of agar-degrading bacteria is prob- 7 days. Single colonies that formed pits or craters were ably best explained by their likely role in the carbon cycle picked and were checked for purity by repeated streaking involving the breakdown of agar and other sulfated galactans on the same medium. One of the colonies exhibiting (Armisen & Galactas, 1987; Pomin, 2010), which form a agarolytic activities was chosen as a representative strain for significant component of the cell walls of red and green algae, further study and was given the designation WH0801T. Cultures were maintained on MA slants at room The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene temperature, and stock cultures were kept in tryptone sequence of strain WH0801T is GQ200591. soya broth (Oxoid) supplemented with 1 % (w/v) NaCl A supplementary table is available with the online version of this paper. (TNB) and 20 % (v/v) glycerol at 270 uC.

019810 Printed in Great Britain 493 Z.-J. Du and others

Phenotypic features of the isolate (i.e. colony morphology, system (Sasser, 1990). Agarivorans albus MKT 106T was pigment production and agar hydrolysis) were determined used for comparison under the same cultural conditions. by cultivating the isolate on MA at 25 uC. Motility was Phenotypic examination of strain WH0801T suggested that assessed in a semi-solid medium prepared according to it belonged to the genus Agarivorans, as cells were Gram- Barrow & Feltham (2004). The tube was incubated at 25 uC stain-negative, strictly aerobic, motile by single polar flagella, for 5 days. Flagellation was observed with a light micro- and agar-hydrolysing. Spores were not formed. No growth scope after staining with Ryu staining solution (West et al., occurred without NaCl. Moreover, strain WH0801T could 1977). Growth with 0, 0.5, 1, 3, 5, 7 and 9 % (w/v) NaCl be readily distinguished from Agarivorans albus by the was assessed on appropriately modified plate count agar production of a light yellow pigment and by its ability to (PCA; Oxoid). Inoculated plates were incubated at 25 uC produce esterase (C4), esterase lipase (C8) and b-glucosi- for up to 5 days. The effects of different temperatures on dase. Comparison of strain WH0801T with Agarivorans growth were assessed on tryptone soya agar (TSA; Oxoid) albus MKT 106T is given in Table 1. The complete plates supplemented with 2.0 % (w/v) NaCl and incubated morphological and biochemical data for strain WH0801T at 4, 10, 15, 28, 30, 37, 42, 45 and 50 uC. The pH range for are given in the species description. growth was determined for the culture in marine broth 2216 at various pH, adjusted with HCl or NaOH (1 M). The almost-complete 16S rRNA gene sequence (1455 nt) of strain WH0801T was obtained and used to determine the Catalase and oxidase activity were examined by standard phylogenetic placement of strain WH0801T (Fig. 1). The methods as described by La´nyı´ (1987). The API 20E, API 16S rRNA gene sequence comparison showed clearly that 20NE and API 50CH tests (bioMe´rieux) were used strain WH0801T is a member of the genus Agarivorans. The according to the manufacturer’s instructions, except that organism showed the highest sequence similarity (99.1 %) the inoculum was prepared by suspending cells in a 2 % with Agarivorans sp. LQ48 (Long et al., 2010). However, (w/v) NaCl solution. The API 50 CH strips were read after the sequence similarity observed between the new isolate 7 days of incubation at 25 uC. Enzyme activities were and its closest relative, Agarivorans albus, was 96.1 %, examined by using the API ZYM test (bioMe´rieux). All API which is below the threshold value (97 %) used for defining tests were performed in duplicate and Agarivorans albus bacterial species as proposed by Stackebrandt & Goebel MKT 106T was used for comparison under the same (1994). On the other hand, the similarity between strain cultural conditions. WH0801T and the type strains of Aliagarivorans marinus Antibiotic sensitivity was assessed as follows: a cell and Aliagarivorans taiwanensis were 93.6 % and 93.2 %, suspension (~107 cells ml21) was swabbed over the surface respectively. Significantly lower levels of similarity were of Iso-Sensitest agar (Oxoid) plates supplemented with 3 % shown with species in other genera (less than 93 % (w/v) NaCl to create a uniform lawn before aseptic similarity). The DNA–DNA relatedness value between placement of antibiotic discs onto the agar surface. The strain WH0801T and Agarivorans albus MKT 106T was inoculated plates were incubated overnight at 28 uC. 33.9±0.85 % (experiment repeated twice). Therefore these two strains are not related at the species level, since the DNA was extracted and purified by using a genomic DNA extraction kit (Sangon, China). The gene encoding 16S rRNA was amplified by PCR with two universal primers, Table 1. Comparison of strain WH0801T with Agarivorans 27f and 1492r (Jordan et al., 2007). The 16S rRNA gene albus MKT 106T sequence of strain WH0801T was submitted to GenBank T T and similar sequences were searched in public databases Strains: 1, strain WH0801 ,2,Agarivorans albus MKT 106 . + using the BLAST algorithm. The phylogenetic placement of , Positive; 2, negative. Data from this study. strain WH0801T and several closely related species was investigated using 16S rRNA gene sequence data; a tree was Characteristic 1 2 reconstructed by using the neighbour-joining method Pigment of colony Light yellow White implemented in the computer program MEGA version 4.1 Gelatin hydrolysis 2 + (Tamura et al., 2007) and statistical reliability was assessed Utilization of: from 1000 bootstrap pseudoreplicates. The G+C content Mannitol 2 + T of DNA isolated from strain WH0801 was determined N-Acetylglucosamine 2 + using the thermal denaturation method (Marmur & Doty, Maltose 2 + 1962). DNA–DNA hybridization between strain WH0801T Acid from: and Agarivorans albus MKT 106T was carried out by Sucrose + 2 applying the optical renaturation method (Huß et al., Amygdalin + 2 1983) under optimal hybridization conditions. L-Arabinose + 2 Production of: Fatty acids from whole cells grown on marine agar 2216 Esterase (C4) + 2 medium at 28 uC for 24 h were extracted, saponified and Esterase lipase (C8) + 2 esterified; this was followed by GC analysis of the fatty acid b-Glucosidase + 2 methyl esters according to the instructions of the MIDI

494 International Journal of Systematic and Evolutionary Microbiology 61 Agarivorans gilvus sp. nov.

Fig. 1. Phylogeny of strain WH0801T and other members of the class Gammaproteobacteria based on 16S rRNA gene sequence similarity. Tree reconstructed using the neighbour-joining method. Numbers along branches represent bootstrap values; only values greater than 50 % are shown. GenBank accession numbers for the 16S rRNA gene sequences used in tree reconstruction are given in parentheses next to each taxon. Bar, 1 % sequence divergence. value falls below the threshold (70 %) recommended for chymotrypsin, a-galactosidase, b-glucuronidase, a-glucosi- species definition by Wayne et al. (1987). dase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase are not produced. The following substrates Therefore, on the basis of phylogenetic, chemotaxonomic are utilized as sole carbon sources: L-arabinose, D-xylose, and other taxonomic data resulting from this study, we D-galactose, D-mannose, arbutin, aesculin ferric citrate, found that strain WH0801T is clearly different from salicin, lactose, starch, glycogen and gentiobiose. The Agarivorans albus MKT 106T. Thus, the novel species following substrates are not utilized as sole carbon sources: Agarivorans gilvus sp. nov. is proposed with strain glycerol, erythritol, D-arabinose, D-ribose, L-xylose, WH0801T as the type strain. D-adonitol, methyl b-D-xylopyranoside, D-glucose, D-fruct- ose, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, Description of Agarivorans gilvus sp. nov. D-sorbitol, methyl a-D-mannopyranoside, methyl a-D- Agarivorans gilvus (gil9vus. L. masc. adj. gilvus pale glucopyranoside, N-acetylglucosamine, amygdalin, cello- yellow, referring to the pale yellow pigmentation of the biose, maltose, melibiose, sucrose, trehalose, inulin, bacterium). melezitose, raffinose, xylitol, turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, potassium gluco- Cells are Gram-stain-negative, non-spore-forming and nate, potassium 2-ketogluconate and potassium 5-ketoglu- rod-shaped, motile by single polar flagella. Colonies are conate. Acids are produced from amygdalin, L-arabinose, pale yellow, smooth and 2.5–3.5 mm in diameter after 48 h D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, of incubation at 28 uC on MA. Strictly aerobic and agar- D-mannitol, N-acetylglucosamine, aesculin ferric citrate, hydrolysing; shallow craters with a clear zone (6–8 mm in salicoside, cellobiose, maltose, lactose, sucrose, starch, diameter) are observed surrounding the colonies due to glycogen and gentiobiose. Resistant to sulfamethoxazole/ agar hydrolysis. Grows at pH 6.5–9.5, optimum at pH 8.4– trimethoprim (23.75/1.25 mg), tetracycline (30 mg), clin- 8.6. Cells grow in the presence of 0.5–5 % (w/v) NaCl and damycin (30 mg), carbenicillin (100 mg), acetylspiramycin at 10–45 uC. Prolific growth occurs at 28–32 uC in media (30 mg), tobramycin (30 mg) and midecamycin (30 mg), containing 3 % (w/v) NaCl. No growth occurs without but sensitive to nalidixic acid (30 mg), gentamicin (120 mg), NaCl. Cells are oxidase, catalase, ONPG and Voges– chloramphenicol (30 mg) and streptomycin (25 mg). Major Proskauer positive. No citrate utilization, H S production, 2 whole-cell fatty acids are C v7c/iso-C 2-OH, C , arginine dihydrolase or indole production. Aesculin and 16 : 1 15:0 16 : 0 C v7c,C ,C 3-OH/iso-C IandC (Supple- starch are hydrolysed, but urea and gelatin are not. Nitrate 18 : 1 12 : 0 14 : 0 16 : 1 14 : 0 mentary Table S1 available in IJSEM Online). The genomic is not reduced to nitrite. Activity is detected for alkaline DNA G+C content of the type strain is 48.5 mol%. phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acidic phosphatase, The type strain is WH0801T (5NRRL B-59247T 5CGMCC naphthol-AS-BI-phosphohydrolase, b-galactosidase and 1.10131T), isolated from the surface of seaweed collected in b-glucosidase. Lipase (C14), cystine arylamidase, trypsin, the shallow coastal region of Weihai, China. http://ijs.sgmjournals.org 495 Z.-J. Du and others

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